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Abstract:

Methods are provided to increase resistance to cell damage in a subject.
The increase in resistance to cell damage in a subject in the subject is
accomplished by decreasing activity of eEF2 kinase in the subject. The
eEF2 kinase activity can be decreased by decreasing the amount of
functional eEF2 kinase produced by the subject, including contacting the
eEF2 kinase with a compound that inhibits phosphorylation of eEF2 kinase
substrate or decreasing the amount of functional eEF2 kinase is decreased
by reducing expression of a gene encoding the eEF2 kinase.

Claims:

1. A method of increasing resistance to cell damage in a subject caused
by a cytotoxic agent, the method comprising identifying a subject that
has been, or is suspected of having been, or is expected to be, exposed
to a cytotoxic agent, administering to the subject an amount of a
compound that decreases phosphorylation of an eEF2 kinase substrate by an
eEF2 kinase, wherein said amount is effective to increase protein
turnover in a cell of the subject at a rate effective to increase
resistance to cell damage.

2. The method of claim 1, wherein said phosphorylation of said eEF2
kinase substrate is decreased by decreasing eEF2 kinase catalytic
activity in said cell.

3. The method of claim 2, wherein said eEF2 kinase catalytic activity is
decreased by contacting the eEF2 kinase with the compound.

4. The method of claim 4, wherein said phosphorylation of the eEF2 kinase
substrate is decreased by reducing expression of a gene encoding the eEF2
kinase in said cell.

5. The method of claim 4, wherein said expression of the gene encoding
the eEF2 kinase is reduced by altering said gene such that the gene
encodes a dysfunctional or non-functional eEF2 kinase.

6. The method of claim 4, wherein said expression of the gene encoding
the eEF2 kinase is reduced by contacting said gene, or an mRNA
transcribed from said gene, with the compound, the compound comprising a
polynucleotide selected from the group consisting of an antisense
oligonucleotide, a ribozyme, an siRNA, and an shRNA.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patent
application Ser. No. 11/615,690, filed Dec. 22, 2006, which is a
continuation-in-part of International Patent Application Serial No.
PCT/US2005/022741, filed Jun. 24, 2005, which claims priority to U.S.
Provisional Patent Application Ser. No. 60/582,411, filed on Jun. 24,
2004. U.S. patent application Ser. No. 11/615,690 also claims priority
under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No.
60/819,688, which was filed on Jul. 10, 2006. The disclosures of all of
the foregoing applications are hereby incorporated by reference in their
entireties.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of cell damage and the
development of compositions and methods to increase resistance to cell
damage. In particular, the invention relates to the manipulation of the
elongation factor 2 (eEF2) kinase in order to increase resistance to cell
damage and increase life span of cells.

BACKGROUND OF THE INVENTION

[0004] Accumulation of damaged cellular proteins is postulated to be a
major contributor to aging and sensitivity to cell damage. Decreases in
both protein synthesis and degradation rates may result in the
persistence of defective or modified proteins and thus the overall rate
of protein turnover can affect the cells' response to cell damage.

[0006] To uncover the physiological role of eEF2K eEF2K knockout mice are
prepared. Despite a complete lack of eEF2K activity, eEF2K knockout mice
have normal development, behavior and reproduction. Moreover, these mice
have increased lifespan. However, fibroblasts from eEF2K knockout mice
are found to be resistant to various cytotoxic agents. The effect of
eEF2K knockout on cell resistance to cytotoxic agents may depend on
functional p53 since it is abolished in cells in which p53 is
inactivated. Intriguingly, knockout mice have significantly extended
maximal lifespan. These findings suggest that eEF2K is a modulator of
stress resistance and aging, and that its inactivation could protect
cells from stress-induced injury and increase life span in mammals.

[0007] One of the hallmarks of aging is the progressive decline in the
rate of protein synthesis and degradation. This decline in protein
turnover can be a major factor contributing to an increase in the
concentration of damaged proteins with age. Therefore, by regulating the
overall rate of protein synthesis and/or degradation it might be possible
to modulate the rate of aging. Since knockout of eEF2 kinase increases
maximal life span in mice, manipulation of expression or activity if eEF2
kinase may offer a therapeutic basis for regulating protein turnover and
reducing cell damage due to stress, exposure to chemotherapeutic agents,
and other factors.

SUMMARY OF THE INVENTION

[0008] In accordance with the present invention, it has now been shown for
the first time that a decrease in activity of eEF2 kinase causes an
increase in overall protein turnover in cells, the result being increased
resistance to cell damage, particularly stress-induced cell damage. Since
the same enzyme is present in all animals, including humans, it is now
clearly predictable that increasing protein turnover in cells,
particularly through the inhibition of this enzyme, in human and animal
subjects will result in decreasing cell death. The discoveries made in
accordance with the present invention enable a variety of useful methods,
kits and pharmaceutical formulations directed to increasing resistance to
cell damage in the cells of a subject, thereby decreasing cell death.

[0009] According to one aspect of the invention, methods are provided to
increase resistance to cell damage in a subject. These methods comprise
increasing protein turnover in the subject, the increase in protein
turnover resulting in the increased resistance to cell damage of the
subject. Preferably, the protein turnover in the subject is accomplished
by decreasing activity of eEF2 kinase in the subject. In one embodiment,
the eEF2 kinase activity is decreased by decreasing the amount of
functional eEF2 kinase produced by the subject. In a preferred embodiment
the amount of functional eEF2 kinase is decreased by decreasing eEF2
kinase activity in the cell, preferably by contacting the eEF2 kinase
with a compound that inhibits phosphorylation of eEF2. In another
preferred embodiment, the amount of functional eEF2 kinase is decreased
by reducing expression of a gene encoding the eEF2 kinase. Alternatively,
the amount of functional eEF2 kinase is decreased by altering a gene
encoding the eEF2 kinase such that the gene encodes a dysfunctional or
non-functional eEF2 kinase.

[0010] The invention also provides a method of increasing resistance to
cell damage in a subject caused by a cytotoxic agent. The method includes
identifying a subject that has been, or is suspected of having been, or
is expected to be, exposed to a cytotoxic agent; administering to the
subject an amount of a compound that decreases phosphorylation of an eEF2
kinase substrate by an eEF2 kinase. The amount is effective to increase
protein turnover in a cell of the subject at a rate effective to increase
resistance to cell damage. In one embodiment, the phosphorylation of the
eEF2 kinase substrate is decreased by decreasing eEF2 kinase catalytic
activity in the cell. In another embodiment, the eEF2 kinase catalytic
activity is decreased by contacting the eEF2 kinase with the compound. In
yet another embodiment, the phosphorylation of the eEF2 kinase substrate
is decreased by reducing expression of a gene encoding the eEF2 kinase in
the cell. The expression of the gene encoding the eEF2 kinase can be
reduced by altering the gene such that the gene encodes a dysfunctional
or non-functional eEF2 kinase. Alternatively, the expression of the gene
encoding the eEF2 kinase can be reduced by contacting the gene, or an
mRNA transcribed from the gene, with the compound that contains a
polynucleotide selected from the group consisting of an antisense
oligonucleotide, a ribozyme, an siRNA, and an shRNA. In one example, the
compound has a polynucleotide containing a nucleotide sequence
complementary to a nucleotide sequence encoding a polypeptide comprising
the amino acid sequence of SEQ ID NO: 2, such as a nucleotide sequence
complementary to a nucleotide sequence having the nucleotide sequence of
SEQ ID NO: 1. In the above-disclosed method, the cytotoxic agent can be
selected from the group consisting of treatment increased acidity,
oxidative stress, a chemotherapy agent, ionizing radiation, ultraviolet
radiation, and free radicals. Examples of the chemotherapy agent include
camptothecin (CPT), doxorubicin (DOX), and taxol.

[0011] According to another aspect of the invention, a genetically
manipulated non-human organism is provided, in which an enzyme that
negatively regulates protein synthesis is dysfunctional, non-functional
or absent. Preferably, the organism is a rodent, preferably a mouse and
the enzyme is eEF2 kinase. Another aspect of the present invention
relates to vector constructs useful in the construction of genetically
manipulated non-human organism, in which eEF2 kinase is dysfunctional,
non-functional or absent.

[0012] According to another aspect of the invention, a pharmaceutical
formulation for increasing resistance to cell damage of a subject is
provided. The formulation comprises an agent that increases cellular
protein turnover in a biologically compatible medium. Preferably, the
formulation comprises an inhibitor of eEF2 activity or gene expression.

[0013] Various diagnostic and prognostic assays and kits are also provided
in accordance with the present invention, as described in greater detail
below. Other features and advantages of the present invention will be
understood by reference to the drawings, detailed description and
examples that follow.

[0016] FIG. 3. eEF2 kinase deficiency increases the resistance of mouse
embryonic fibroblasts to chemotherapeutic drugs. a, b. Graphs
representing the response of eEF2+/+ (WT) and eEF2K-/- (KO) MEFs to CPT
and DOX, measured by MTT assay. Cells are incubated for 24 h at indicated
concentrations of a drug. c. Graphs of MTT assay comparing the response
of eEF2K+/+, eEF2K-/- and KO (eEF2K) (cell line, transfected with cDNA of
eEF2K) to doxorubicin. Experiment performed as described in a, b. d. Flow
cytometric analysis of eEF2K+/+ and eEF2K-/- MEFs treated with 600 ng/ml
of DOX for 24 h. e. Analysis of apoptotic DNA fragmentation in wild type
and eEF2K-/- stable cell lines in response to a serum starvation (SS) for
48 h.

[0017] FIG. 4. Effect of eEF2 kinase knockout on drug resistance depends
on functional p53. a. Drug sensitivity assay of eEF2K+/+ and eEF2K-/-
stable cell lines and the same cell lines expressing dominant-negative
p53 mutant GSE56. Cells are treated with indicated concentrations of DOX
and after 24 h cell viability is measured by MTT assay. b. Drug
sensitivity assay of eEF2K+/+ and eEF2K-/- stable cell lines and the same
cell lines treated with PFT a. MTT assay is done as described in a. c.
Western blot analysis of p53, p21 and 13 tubulin expression in wild type
and eEF2K-/- MEFs after DOX treatment. d. Quantification of the amount of
p21 protein before and after incubation with DOX.

[0019] FIG. 6. eEF2K-/- mice are resistant to gamma irradiation. a. The
survival of mice after whole-body gamma irradiation. Mice at 8 to 12
weeks of age are exposed to 8 Gy of whole-body γ-irradiation and
survival is monitored daily. Each cohort contains 10 mice including 5
males and 5 females. b. The appearances of eEF2K+/+ mice (bottom) and
eEF2K-/- mice (top) one month after 8 Gy of γ-irradiation.

[0020] FIG. 7. eEF2K-/- cells are resistant to apoptosis. a.
Phase-contrast images of cells. MEFs are treated with 1.6 μM of
doxorubicin for 24 hours. b. TUNEL assay of MEFs treated with 1.6 μM
of doxorubicin for 12 hours. Apoptotic cells are analyzed by flow
cytometry. c. The effect of introduction of eEF2K cDNA into eEF2K-/- MEFs
on the activation of caspase 3 induced by 0.8 μM of doxorubicin or 600
μM of H2O2. Activation of caspase 3 is analyzed by western
blotting.

[0021] FIG. 8. Phosphorylation of eEF2 occurs in cells undergoing
apoptosis. a. Western blot analysis of phosphorylated eEF2. NIH3T3 cells
are exposed to 80 μM of hydrogen peroxide for the indicated time
periods and eEF2 phosphorylation is analyzed by western blotting using
antibodies specific for phosphorylated eEF2. b. Immunostaining of
phosphorylated eEF2 in NIH3T3 cells. Cells are exposed to 400 μM of
H2O2 for 3 hours and phosphorylated eEF2 is detected by
immunostaining in both H2O2-treated and untreated (UT) cells.
Cells showing the highest phosphorylated eEF2 levels are indicated by the
white arrowheads. c. HeLa cells are treated with 400 μM of
H2O2 for 3 hours and immunostaining is performed. Cells with
condensed chromatin are indicated by gray arrowheads. d,e,f,g.
immunostaining of phosphorylated eEF2 in human lymph nodes and brain. e.
higher magnification of d.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is based in part on the discovery that
decreasing eEF2 kinase activity in a cell results in an increase in
resistance to damage to the cell, as well as a resistance to programmed
cell death (apoptosis). Without being bound by any particular theory, it
is believed that the increased levels of protein translation and protein
turnover that result from a decrease in eEF2 kinase activity help protect
cells from damage and subsequent death. The term "protein turnover" is
art-recognized and refers to the coordinated synthesis and degradation of
proteins that occurs in living cells, tissues and organisms.

Cell Death

[0023] Apoptosis is referred to as a process of "programmed cell death."
During normal somatic development, cell populations in specific organs or
tissues may be programmed for death as part of the developmental
progression of tissue remodeling or obsolescence. See J. J. Cohen, Avd.
Immunol. 50:55-85 (1991); M. Baring a, Science 259:762-3 (1993).
Apoptosis is internally triggered by biochemical or biomolecular
mechanisms intrinsic to the cell cycle, resulting in an activation of
endogenous endonucleases (enzymes that degrade DNA), leading to DNA
strand breaks between nucleosomes and degradation of the genomic DNA by
fragmentation. A. H. Wyllie, Nature 284:555-6 (1980). Apoptosis in mature
tissues occurs in normal processes such as inflammation or rejuvenation.
M. Schmied et al., Am. J. Pathol. 143:446-52 (1993); Abnormal clonal
proliferations in immunologic diseases or malignancies may be related to
a failure of normal apoptosis. J. Marx, Science 259:760-1 (1993).

[0024] The relationship of apoptosis and/or cell damage to the cell cycle,
including checkpoint controls, during cancer chemotherapy is a subject of
interest to oncologists and molecular biologists. See T. Shimizu et al.,
Cancer Res. 55:228-231 (1995); O'Connor, supra. (1992). The expression of
p53 in damaged cells is one factor in determining the course of divergent
biochemical pathways, which can lead to either DNA repair or apoptosis. E
Yonish-Rouach et al., Mol Cell Biol 13:1415-23 (1993); D E Fisher, Cell
78:539-542 (1994).

[0025] In chemotherapy for malignancy, treatments with targeted cytotoxic
effect have involved a number clinical considerations: they may be used
in the primary effort to control cancer (induction chemotherapy), or as
an adjunct to surgery or radiotherapy (adjuvant chemotherapy). DeVita,
supra (1994). Local treatments have included infusion of a targeted
cytotoxic compound into body cavities to control the spread of
malignancies such as breast or ovarian cancers.

Cell Damage

[0026] Cell damage is caused by a treatment that causes stress to the
cell. In a particularly preferred embodiment. Stress may be caused by a
variety of factors, including increased acidity, oxidative stress, or
exposure of the cell to a compound used for treatment of a disease state,
such as for example, camptothecin (CPT), doxorubicin (DOX), or taxol.
Cell damage may also stem from ionizing radiation, ultraviolet radiation
and free radicals.

Decreasing Kinase Activity

[0027] The present invention relates to a method of reducing damage to a
cell or increasing resistance to damage to a cell, comprising decreasing
eEF2 kinase activity in the cell. The cell may be in cultures, in a
tissue or in a subject in need of treatment. The subject may be a mammal
such as a human.

[0028] The term "inhibition" refers to the reduction or down regulation of
a process or activity that results in the absence or minimization of that
process or activity. The term "inhibit" or "inhibiting", in relationship
to the term "activity" means that an activity is decreased or prevented
in the presence of a compound as opposed to in the absence of the
compound.

[0029] In a preferred embodiment, the decrease in eEF2 kinase activity, is
accomplished by contacting the eEF2 kinase with a compound that decreases
phosphorylation of eEF2 by the eEF2 kinase.

[0030] The term "contact" or "contacting" means bringing at least two
moieties together, whether in an in vitro system or an in vivo system.

Compounds

[0031] The term "compound" is used herein in the context of a "test
compound" or a "drug candidate compound" described in connection with the
assays of the present invention. As such, these compounds comprise
organic or inorganic compounds, derived synthetically or from natural
sources. The compounds include inorganic or organic compounds such as
polynucleotides, lipids or hormone analogs that are characterized by
relatively low molecular weights. Other biopolymeric organic test
compounds include peptides comprising from about 2 to about 40 amino
acids and larger polypeptides comprising from about 40 to about 500 amino
acids, such as antibodies or antibody conjugates.

Assays to Identify Compounds

[0032] There are a variety of methods that may be used to identify
compounds capable of inhibition of the activity of eEF2 kinase. The
affinity of the compounds to eEF2 kinase may be determined in an
experiment that detects changed reaction conditions after phosphorylation
of eEF2. eEF2 kinase is incubated with eEF2 and ATP in an appropriate
buffer. The combination of these components results in the in vitro
phosphorylation of eEF2. Sources of compounds include any commercially
available screening library, peptides in a phage display library or an
antibody fragment library, and compounds that have been demonstrated to
have binding affinity for eEF2 kinase.

[0033] The term "binding affinity" is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively, (such as "strong", "weak", "high", or "low") or
quantitatively (such as measuring the KD).

[0034] eEF2 kinase can be prepared in a number of ways depending on
whether the assay will be run using cells, cell fractions or
biochemically, on purified protein. eEF2 kinase can be applied as
complete a polypeptides or as a polypeptide fragment, which still
comprises eEF2 kinase catalytic activity.

[0035] The term "assay" means any process used to measure a specific
property of a compound. A "screening assay" means a process used to
characterize or select compounds based upon their activity from a
collection of compounds.

[0036] The term "polypeptide" relates to proteins, proteinaceous
molecules, fractions of proteins peptides and oligopeptides.

[0037] Identification of small molecules inhibiting the activity of the
eEF2 kinase is performed by measuring changes in levels of phosphorylated
eEF2 kinase substrate, which can be a peptide or a full-length protein,
or ATP. A preferred substrate is eEF2. Since ATP is consumed during the
phosphorylation of eEF2 kinase substrate, its levels correlate with the
kinase activity. Measuring ATP levels via chemiluminescent reactions
therefore represents a method to measure kinase activity in vitro (Perkin
Elmer). In a second type of assay, changes in the levels of
phosphorylated eEF2 kinase substrate are detected with phosphospecific
agents and are correlated to eEF2 kinase activity. These levels are
detected in solution or after immobilization of the substrate on a
microtiter plate or other carrier. In solution, the phosphorylated eEF2
kinase substrate is detected via fluorescence resonance energy transfer
(FRET) between the Eu labeled substrate and an APC labeled
phosphospecific antibody (Perkin Elmer), via fluorescence polarization
(FP) after binding of a phosphospecific antibody to the fluorescently
labeled phosphorylated eEF2 kinase substrate, via an Amplified
Luminescent Proximity Homogeneous Assay (ALPHA) using the phosphorylated
eEF2 kinase substrate and phosphospecific antibody, both coupled to ALPHA
beads (Perkin Elmer) or using the IMAP binding reagent that specifically
detects phosphate groups and thus alleviates the use of the
phosphospecific antibody (Molecular Devices). Alternatively, the eEF2
kinase substrate is immobilized directly or by using biotin-streptavidin
on a microtiter plate. After immobilization, the level of phosphorylated
eEF2 kinase substrate is detected using a classic ELISA where binding of
the phosphospecific antibody is either monitored via an enzyme such as
horseradish peroxidase (HRP) or alkaline phosphatase (AP) which are
either directly coupled to the phosphospecific antibody or are coupled to
a secondary antibody. Enzymatic activity correlates to phosphorylated
eEF2 kinase substrate levels. Alternatively, binding of the Eu-labeled
phosphospecific antibody to the immobilized phosphorylated eEF2 kinase
substrate is determined via time resolved fluorescence energy (TRF)
(Perkin Elmer). In addition, the eEF2 kinase substrate can be coated on
FLASH plates (Perkin Elmer) and phosphorylation of the eEF2 kinase
substrate is detected using 33P labeled ATP or 125I labeled
phosphospecific antibody.

[0038] The term "agent" means any molecule, including polypeptides,
polynucleotides and small molecules.

[0039] Small molecules are randomly screened or are preselected based upon
drug class, (i.e. known kinase inhibitors), or upon virtual ligand
screening (VLS) results. VLS uses virtual docking technology to test
large numbers of small molecules in silico for their binding to the
polypeptide of the invention. Small molecules are added to the kinase
reaction and their effect on levels of phosphorylated eEF2 is measured
with one or more of the above-described technologies.

[0040] Small molecules that inhibit the kinase activity are identified and
are subsequently tested at different concentrations. IC50 values are
calculated from these dose response curves. Strong binders have an
IC50 in the nanomolar and even picomolar range.

Reduction in eEF2 Gene Expression

[0041] In another preferred embodiment, the present invention relates to a
method of reducing damage to a cell or increasing resistance to damage to
a cell, comprising decreasing eEF2 kinase activity by reducing the
expression of a gene encoding the eEF2 kinase. This reduction in
expression can be accomplished by a variety of methods and in preferred
embodiments it is accomplished by altering the gene such that the gene
encodes a dysfunctional or non-functional eEF2 kinase.

[0042] The term "expression" comprises both endogenous expression and
overexpression by transduction.

[0043] A variety of means are available for altering a gene to effect
expression. In a special embodiment the expression of a gene encoding the
eEF2 kinase is reduced by contacting the gene, or an mRNA transcribed
from the gene, with a compound comprising a polynucleotide selected from
the group consisting of an antisense oligonucleotide, a ribozyme, a small
interfering RNA (siRNA), and a short hairpin RNA (shRNA). In certain
embodiments the compound comprises a polynucleotide comprising a
nucleotide sequence complementary to a nucleotide sequence encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (eEF2
kinase polypeptide sequence). In a particularly preferred embodiment the
compound comprises a nucleotide sequence complementary to a nucleotide
sequence comprising the nucleotide sequence of SEQ ID NO: 1 (eEF2 kinase
polynucleotide sequence).

[0044] The term "polynucleotide" means a polynucleic acid, in single or
double stranded form, and in the sense or antisense orientation,
complementary polynucleic acids that hybridize to a particular
polynucleic acid under stringent conditions, and polynucleotides that are
homologous in at least about 60 percent of its base pairs, and more
preferably 70 percent of its base pairs are in common, most preferably 90
percent, and in a special embodiment 100 percent of its base pairs. The
polynucleotides include polyribonucleic acids, polydeoxyribonucleic
acids, and synthetic analogues thereof. The polynucleotides are described
by sequences that vary in length, that range from about 10 to about 5000
bases, preferably about 100 to about 4000 bases, more preferably about
250 to about 2500 bases. A preferred polynucleotide embodiment comprises
from about 10 to about 30 bases in length. A special embodiment of
polynucleotide is the polyribonucleotide of from about 10 to about 22
nucleotides, more commonly described as small interfering RNAs (siRNAs).
Another special embodiment are nucleic acids with modified backcartilages
such as peptide nucleic acid (PNA), polysiloxane, and
2'-O-(2-methoxy)ethylphosphorothioate, or including non-naturally
occurring nucleic acid residues, or one or more nucleic acid
substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,
amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter
molecule to facilitate its detection.

[0045] The term "antisense nucleic acid" refers to an oligonucleotide that
has a nucleotide sequence that interacts through base pairing with a
specific complementary nucleic acid sequence involved in the expression
of the target such that the expression of the gene is reduced.
Preferably, the specific nucleic acid sequence involved in the expression
of the gene is a genomic DNA molecule or mRNA molecule that encodes (a
part of) the gene. This genomic DNA molecule can comprise regulatory
regions of the gene, or the coding sequence for the mature gene.

[0046] The term `complementary to a nucleotide sequence` in the context of
antisense oligonucleotides and methods should be understood as
sufficiently complementary to such a sequence as to allow hybridization
to that sequence in a cell, i.e., under physiological conditions.

[0047] The term "hybridization" means any process by which a strand of
nucleic acid binds with a complementary strand through base pairing. The
term "hybridization complex" refers to a complex formed between two
nucleic acid sequences by virtue of the formation of hydrogen bonds
between complementary bases. A hybridization complex may be formed in
solution (e.g., C0t or R0t analysis) or formed between one nucleic acid
sequence present in solution and another nucleic acid sequence
immobilized on a solid support (e.g., paper, membranes, filters, chips,
pins or glass slides, or any other appropriate eEF2 to which cells or
their nucleic acids have been fixed). The term "stringent conditions"
refers to conditions that permit hybridization between polynucleotides
and the claimed polynucleotides. Stringent conditions can be defined by
salt concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art. In
particular, reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization temperature can
increase stringency.

Antisense

[0048] The down regulation of gene expression using antisense nucleic
acids can be achieved at the translational or transcriptional level using
an expression-inhibitory agent. Antisense nucleic acids of the invention
are preferably nucleic acid fragments capable of specifically hybridizing
with all or part of a nucleic acid encoding a eEF2 kinase or the
corresponding messenger gene or mRNA. In addition, antisense nucleic
acids may be designed which decrease expression of the nucleic acid
sequence capable of encoding a eEF2 kinase by inhibiting splicing of its
primary transcript. Any length of antisense sequence is suitable for
practice of the invention so long as it is capable of down-regulating or
blocking expression of a nucleic acid coding for eEF2 kinase. Preferably,
the antisense sequence is at least about 17 nucleotides in length. The
preparation and use of antisense nucleic acids, DNA encoding antisense
RNAs and the use of oligo and genetic antisense is known in the art. \

[0049] The term "expression inhibitory agent" means a polynucleotide
designed to interfere selectively with the transcription, translation
and/or expression of a specific polypeptide or protein normally expressed
within a cell. More particularly, "expression inhibitory agent" comprises
a DNA or RNA molecule that contains a nucleotide sequence identical to or
complementary to at least about 17 sequential nucleotides within the
polyribonucleotide sequence coding for a specific polypeptide or protein.
Exemplary expression inhibitory molecules include ribozymes, double
stranded siRNA molecules, self-complementary single-stranded siRNA
molecules, genetic antisense constructs, and synthetic RNA antisense
molecules with modified stabilized backbones.

[0050] One embodiment of expression-inhibitory agent is a nucleic acid
that is antisense to a nucleic acid comprising SEQ ID NO: 1. For example,
an antisense nucleic acid (e.g. DNA) may be introduced into cells in
vitro, or administered to a subject in vivo, as gene therapy to inhibit
cellular expression of nucleic acids comprising SEQ ID NO: 1. Antisense
oligonucleotides preferably comprise a sequence containing from about 17
to about 100 nucleotides and more preferably the antisense
oligonucleotides comprise from about 18 to about 30 nucleotides.
Antisense nucleic acids may be prepared from about 10 to about 30
contiguous nucleotides complementary to a nucleic acid sequence selected
from the sequences of SEQ ID NO: 1.

[0051] The antisense nucleic acids are preferably oligonucleotides and may
consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides,
or some combination of both. The antisense nucleic acids can be synthetic
oligonucleotides. The oligonucleotides may be chemically modified, if
desired, to improve stability and/or selectivity. Since oligonucleotides
are susceptible to degradation by intracellular nucleases, the
modifications can include, for example, the use of a sulfur group to
replace the free oxygen of the phosphodiester bond. This modification is
called a phosphorothioate linkage. Phosphorothioate antisense
oligonucleotides are water soluble, polyanionic, and resistant to
endogenous nucleases. In addition, when a phosphorothioate antisense
oligonucleotide hybridizes to its mRNA target, the RN202-315NA duplex
activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the
mRNA component of the hybrid molecule.

[0052] In addition, antisense oligonucleotides with phosphoramidite and
polyamide (peptide) linkages can be synthesized. These molecules should
be very resistant to nuclease degradation. Furthermore, chemical groups
can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5)
of pyrimidines to enhance stability and facilitate the binding of the
antisense oligonucleotide to its TARGET site. Modifications may include
2'-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy
phosphorothioates, modified bases, as well as other modifications known
to those of skill in the art.

Ribozyme

[0053] Another type of expression-inhibitory agent that reduces the levels
of mRNA is the ribozyme. Ribozymes are catalytic RNA molecules (RNA
enzymes) that have separate catalytic and substrate binding domains. The
substrate binding sequence combines by nucleotide complementarity and,
possibly, non-hydrogen bond interactions with its mRNA sequence. The
catalytic portion cleaves the mRNA at a specific site. The substrate
domain of a ribozyme can be engineered to direct it to a specified mRNA
sequence. The ribozyme recognizes and then binds eEF2 kinase mRNA through
complementary base pairing. Once it is bound to the correct eEF2 kinase
mRNA site, the ribozyme acts enzymatically to cut the eEF2 kinase mRNA.
Cleavage of the mRNA by a ribozyme destroys its ability to direct
synthesis of the corresponding polypeptide. Once the ribozyme has cleaved
its eEF2 kinase mRNA sequence, it is released and can repeatedly bind and
cleave at other mRNAs. Ribozyme forms include a hammerhead motif, a
hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in
association with an RNA guide sequence) motif or Neurospora VS RNA motif.
Ribozymes possessing a hammerhead or hairpin structure are readily
prepared since these catalytic RNA molecules can be expressed within
cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res.
20:4581-9). A ribozyme of the present invention can be expressed in
eukaryotic cells from the appropriate DNA vector.

[0054] If desired, the activity of the ribozyme may be augmented by its
release from the primary transcript by a second ribozyme (Ventura, et al.
(1993) Nucleic Acids Res. 21:3249-55).

[0055] The term "vectors" relates to plasmids as well as to viral vectors,
such as recombinant viruses, or the nucleic acid encoding the recombinant
virus.

[0056] Ribozymes may be chemically synthesized by combining an
oligodeoxyribonucleotide with a ribozyme catalytic domain (20
nucleotides) flanked by sequences that hybridize to the eEF2 kinase mRNA
after transcription. The oligodeoxyribonucleotide is amplified by using
the substrate binding sequences as primers. The amplification product is
cloned into a eukaryotic expression vector.

[0057] Ribozymes are expressed from transcription units inserted into DNA,
RNA, or viral vectors. Transcription of the ribozyme sequences are driven
from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase
II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or
pol III promoters will be expressed at high levels in all cells; the
levels of a given pol II promoter in a given cell type will depend on
nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters
are also used, providing that the prokaryotic RNA polymerase enzyme is
expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids
Res. 21:2867-72). It has been demonstrated that ribozymes expressed from
these promoters can function in mammalian cells (Kashani-Sabet, et al.
(1992) Antisense Res. Dev. 2:3-15).

siRNA

[0058] A particularly preferred inhibitory agent is a small interfering
RNA (siRNA). siRNA, preferably short hairpin RNA (shRNA), mediate the
post-transcriptional process of gene silencing by double stranded RNA
(dsRNA) that is homologous in sequence to the silenced RNA. siRNA
according to the present invention comprises a sense strand of 17-25
nucleotides complementary or homologous to a contiguous 17-25 nucleotide
sequence selected from the group of sequences encoding SEQ ID NO: 2,
preferably from SEQ ID NO: 1, and an antisense strand of 17-23
nucleotides complementary to the sense strand. The most preferred siRNA
comprises sense and anti-sense strands that are 100 percent complementary
to each other and the eEF2 kinase polynucleotide sequence. Preferably the
siRNA further comprises a loop region linking the sense and the antisense
strand. A self-complementing single stranded siRNA molecule
polynucleotide according to the present invention comprises a sense
portion and an antisense portion connected by a loop region linker. The
loop can be any length but is preferably 4-30 nucleotides long.
Self-complementary single stranded siRNAs form hairpin loops and are more
stable than ordinary dsRNA. In addition, they are more easily produced
from vectors.

[0059] Analogous to antisense RNA, the siRNA can be modified to confirm
resistance to nucleolytic degradation, or to enhance activity, or to
enhance cellular distribution, or to enhance cellular uptake, such
modifications may consist of modified internucleoside linkages, modified
nucleic acid bases, modified sugars and/or chemical linkage the siRNA to
one or more moieties or conjugates.

[0060] The present invention also relates to compositions, and methods
using said compositions, comprising a DNA expression vector capable of
expressing a polynucleotide capable of increasing resistance to cell
damage and is described hereinabove as an expression inhibition agent.

Intracellular Binding Protein

[0061] A special aspect of these compositions and methods relates to the
down-regulation or blocking of the expression of a eEF2 kinase by the
induced expression of a polynucleotide encoding an intracellular binding
protein that is capable of selectively interacting with the eEF2 kinase
polypeptide. An intracellular binding protein includes any protein
capable of selectively interacting, or binding, with the polypeptide in
the cell in which it is expressed and neutralizing the function of the
polypeptide. Preferably, the intracellular binding protein is a
neutralizing antibody or a fragment of a neutralizing antibody having
binding affinity to an epitope of the eEF2 kinase of SEQ ID NO: 2. More
preferably, the intracellular binding protein is a single chain antibody.

[0062] The term "binding affinity" is a property that describes how
strongly two or more compounds associate with each other in a
non-covalent relationship. Binding affinities can be characterized
qualitatively, (such as "strong", "weak", "high", or "low") or
quantitatively (such as measuring the KD).

[0063] A special embodiment of this composition comprises the
expression-inhibiting agent selected from the group consisting of
antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that
cleaves the polyribonucleotide coding for SEQ ID NO: 2, and a small
interfering RNA (siRNA) that is sufficiently homologous to a portion of
the polyribonucleotide coding for SEQ ID NO: 2, such that the siRNA
interferes with the translation of the eEF2 kinase polyribonucleotide to
the eEF2 kinase polypeptide.

[0064] The polynucleotide expressing the expression-inhibiting agent is
preferably included within a vector. The polynucleic acid is operably
linked to signals enabling expression of the nucleic acid sequence and is
introduced into a cell utilizing, preferably, recombinant vector
constructs, which will express the antisense nucleic acid once the vector
is introduced into the cell. A variety of viral-based systems are
available, including adenoviral, retroviral, adeno-associated viral,
lentiviral, herpes simplex viral or a sendaviral vector systems, and all
may be used to introduce and express polynucleotide sequence for the
expression-inhibiting agents in eEF2 kinase-expressing cells.

[0065] The term "operably linked" or "operably inserted" means that the
regulatory sequences necessary for expression of the coding sequence are
placed in a nucleic acid molecule in the appropriate positions relative
to the coding sequence so as to enable expression of the coding sequence.
This same definition is sometimes applied to the arrangement other
transcription control elements (e.g. enhancers) in an expression vector.
Transcriptional and translational control sequences are DNA expression
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression of a
coding sequence in a host cell.

[0066] The terms "promoter", "promoter region" or "promoter sequence"
refer generally to transcriptional regulatory regions of a gene, which
may be found at the 5' or 3'side of the coding region, or within the
coding region, or within introns. Promoters that may be used in the
expression vectors of the present invention include both constitutive
promoters and regulated (inducible) promoters.

[0067] Typically, a promoter is a DNA regulatory region capable of binding
RNA polymerase in a cell and initiating transcription of a downstream
(3'direction) coding sequence. The typical 5'promoter sequence is bounded
at its 3'terminus by the transcription initiation site and extends
upstream (5'direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence is a transcription initiation
site (conveniently defined by mapping with nuclease S1), as well as
protein binding domains (consensus sequences) responsible for the binding
of RNA polymerase.

[0068] In a preferred embodiment, the viral element is derived from an
adenovirus. Other embodiments of the present invention use retroviral
vector systems which can be constructed from different types of
retrovirus, such as, MoMuLV ("murine Moloney leukemia virus" MSV ("murine
Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus"); SNV ("spleen
necrosis virus"); RSV ("Rous sarcoma virus") and Friend virus. Lentiviral
vector systems may also be used in the practice of the present invention.
In other embodiments of the present invention, adeno-associated viruses
("AAV") are utilized.

[0069] Preferably, the viral vectors used in the methods of the present
invention are replication defective. Such replication defective vectors
will usually pack at least one region that is necessary for the
replication of the virus in the infected cell. These regions can either
be eliminated (in whole or in part), or be rendered non-functional by any
technique known to a person skilled in the art. These techniques include
the total removal, substitution, partial deletion or addition of one or
more bases to an essential (for replication) region. Such techniques may
be performed in vitro (on the isolated DNA) or in situ, using the
techniques of genetic manipulation or by treatment with mutagenic agents.
Preferably, the replication defective virus retains the sequences of its
genome, which are necessary for encapsidating, the viral particles.

[0070] In the vector construction, the polynucleotide agents of the
present invention may be linked to one or more regulatory regions.
Selection of the appropriate regulatory region or regions is a routine
matter, within the level of ordinary skill in the art. Regulatory regions
include promoters, and may include enhancers, suppressors, etc.

[0071] Additional vector systems include the non-viral systems that
facilitate introduction of polynucleotide agents into a patient. For
example, a DNA vector encoding a desired sequence can be introduced in
vivo by lipofection. Synthetic cationic lipids designed to limit the
difficulties encountered with liposome-mediated transfection can be used
to prepare liposomes for in vivo transfection of a gene encoding a marker
(Feigner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7); see
Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et
al. (1993) Science 259:1745-8). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Feigner and Ringold,
(1989) Nature 337:387-8). Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in International
Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No.
5,459,127. The use of lipofection to introduce exogenous genes into the
specific organs in vivo has certain practical advantages and directing
transfection to particular cell types would be particularly advantageous
in a tissue with cellular heterogeneity, for example, pancreas, liver,
kidney, and the brain. Lipids may be chemically coupled to other
molecules for the purpose of targeting. Targeted peptides, e.g., hormones
or neurotransmitters, and proteins for example, antibodies, or
non-peptide molecules could be coupled to liposomes chemically. Other
molecules are also useful for facilitating transfection of a nucleic acid
in vivo, for example, a cationic oligopeptide (e.g., International Patent
Publication WO 95/21931), peptides derived from DNA binding proteins
(e.g., International Patent Publication WO 96/25508), or a cationic
polymer (e.g., International Patent Publication WO 95/21931).

[0073] In another aspect, the invention relates to a method of treating
and/or preventing a disease characterized by an increase in eEF2 kinase
activity in a patient by administering to the patient a therapeutically
effective amount of a composition comprising a compound that decreases
phosphorylation of eEF2 kinase substrate by eEF2 kinase. The term
"condition" or "disease" means the overt presentation of symptoms (i.e.,
illness) or the manifestation of abnormal clinical indicators (e.g.,
biochemical indicators). Alternatively, the term "disease" refers to a
genetic or environmental risk of or propensity for developing such
symptoms or abnormal clinical indicators. A variety of disease states may
be treated utilizing the methods and compounds of the present invention.
In preferred embodiments the disease state is selected from the group
consisting of hypoxia, anoxia, ischemia, stroke, and neurogenerative
diseases such as Parkinson's or Alzheimer's disease.

[0074] In yet another aspect, the invention relates to a method of
protecting a cell population in a patient from a potential source of cell
damage selected from chemotherapy agents, ionizing radiation, ultraviolet
radiation, and free radicals by administering to the patient a
therapeutically effective amount of a composition including a compound
that decreases phosphorylation of eEF2 kinase substrate by eEF2 kinase.
In particular, the invention relates to a method of protecting normal
tissues during chemotherapy of cancer cells by administering to the
patient a therapeutically effective amount of a composition including a
compound that decreases phosphorylation of eEF2 kinase substrate by eEF2
kinase. In one aspect of the invention, the compound includes a
polynucleotide having a nucleotide sequence complementary to a nucleotide
sequence encoding a polypeptide comprising the amino acid sequence of SEQ
ID NO: 2. In another aspect, the ionizing radiation includes gamma
radiation.

[0075] In another aspect, the present invention relates to a knockout
mouse, wherein the knockout mouse comprises a disruption in an eEF2
kinase gene. In a preferred embodiment, the mouse is heterozygous for the
disruption in the eEF2 kinase gene. In an especially preferred
embodiment, the mouse is homozygous for the disruption in the eEF2 kinase
gene. In a preferred embodiment, the disruption occurs in a region of the
eEF2 kinase gene which encodes a catalytic domain of the eEF2 kinase
polypeptide, preferably the region comprises exon 7 or 8 of the eEF2
kinase gene. In an especially preferred embodiment, the mouse exhibits a
phenotype selected from the group consisting of extension of life span
and resistance to stress-induced cell damage.

[0076] In another aspect, the invention relates to an eEF2 kinase knockout
construct, comprising a portion of an eEF2 kinase gene, wherein a portion
of the eEF2 kinase gene is replaced by a selectable marker. In a
preferred embodiment the selectable marker is a gene which encodes for a
polypeptide selected from the group consisting of thymidine kinase,
neomycin phosphotransferase and hygromycin B phosphotransferase. In an
especially preferred embodiment the portion of the eEF2 kinase gene which
is replaced comprises exon 7.

[0077] In another aspect, the invention relates to a method of producing a
mouse with a targeted disruption in an eEF2 kinase gene. The mouse is
obtained by transfecting a population of embryonic stem cells with a
knockout construct in which comprising a portion of the eEF2 kinase gene
with a portion of the eEF2 kinase gene replaced by a marker; selecting a
transfected embryonic stem cell which expresses the marker; introducing
the transfected ES cell into an embryo of an ancestor of the mouse
allowing the embryo to develop to term to produce a chimeric mouse with
the knockout construct in its germline; and breeding the chimeric mammal,
to produce a heterozygous mouse with a targeted disruption in the eEF2
kinase gene.

[0078] An eEF2 kinase knockout construct is typically prepared by
isolating a portion of the genomic or cDNA eEF2 kinase nucleotide
sequence (usually encoding at least one exon and one intron), and
inserting a marker sequence into the eEF2 kinase sequence. The eEF2
kinase gene or gene fragment to be used in preparing this construct may
be obtained in a variety of ways. Generally, the eEF2 kinase DNA molecule
will be at least about 1 kilobase (kb) in length, and preferably will be
3-4 kb in length, thereby providing sufficient complementary sequence for
recognition with chromosomal DNA (i.e., homologous recombination) when
the knockout construct is introduced into the genomic DNA of the
embryonic stem (ES) cell (discussed below).

[0079] A naturally occurring genomic eEF2 kinase fragment or cDNA molecule
to be used in preparing the knockout construct can be obtained using
methods well known in the art such as those described by Sambrook et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989)). Such methods include, for
example, PCR amplification of a particular DNA sequence using
oligonucleotide primers, or screening a genomic library prepared from
cells or tissues that contain the eEF2 kinase gene with a cDNA probe
encoding at least a portion of the same or a highly homologous eEF2
kinase gene in order to obtain at least a portion of the eEF2 kinase
genomic sequence. Alternatively, if a cDNA sequence is to be used in a
knockout construct, the cDNA may be obtained by screening a cDNA library
(preferably one prepared from tissues or that express eEF2 kinase, where
the tissues or cells are derived from the same or a similar species of
mammal as that to be rendered the knockout mammal) with oligonucleotide
probes, homologous cDNA probes, or antibodies (where the library is
cloned into an expression vector). If a promoter sequence is to be used
in the knockout construct, synthetic DNA probes or primers can be
designed for screening a genomic library or for amplification using PCR,
respectively.

[0080] The eEF2 kinase genomic DNA fragment or eEF2 kinase cDNA molecule
prepared for use in the knockout construct must be generated in
sufficient quantity for genetic manipulation. Amplification may be
conducted by 1) placing the fragment into a suitable vector and
transforming bacterial or other cells that can rapidly amplify the
vector, 2) by PCR amplification, 3) by synthesis with a DNA synthesizer,
or 4) by other suitable methods.

[0081] The eEF2 kinase genomic DNA fragment, cDNA molecule, or PCR
fragment to be used in making the eEF2 kinase knockout construct can be
digested with one or more restriction enzymes selected to cut at a
location(s) such that a second DNA molecule encoding a marker gene can be
inserted in the proper position within the eEF2 kinase genomic DNA
fragment, cDNA molecule, or PCR fragment to be used in the construct. The
proper position for marker gene insertion is one that will serve to
decrease or prevent transcription and/or expression of the full length
endogenous eEF2 kinase gene. This position will depend on various factors
such as the available restriction sites in the sequence to be cut,
whether an exon sequence or a promoter sequence, or both is (are) to be
interrupted, and whether several isoforms of eEF2 kinase exist in the
mammal (due to alternative splicing) and only one such isoform is to be
disrupted. Preferably, the enzyme(s) selected for cutting the eEF2 kinase
genomic DNA, cDNA molecule, or PCR fragment will generate a longer arm
and a shorter arm, where the shorter arm is at least about 300 base pairs
(bp). In some cases, it will be desirable to actually delete a portion or
even all of one or more introns or exons of this native genomic or cDNA
molecule. In these cases, the eEF2 kinase genomic DNA, cDNA molecule, or
PCR fragment can be cut with appropriate restriction endonucleases such
that a fragment of the proper size and proper location can be removed.

[0082] The marker gene used in the knockout construct can be any nucleic
acid molecule that is detectable and/or assayable after it has been
incorporated into the genomic DNA of the ES cell, and ultimately the
knockout mammal, however typically it is an antibiotic resistance gene or
other gene whose expression or presence in the genome can easily be
detected. Preferably, the marker gene encodes a polypeptide that does not
naturally occur in the mammal. The marker gene is usually operably linked
to its own promoter or to another strong promoter such as the thymidine
kinase (TK) promoter or the phosphoglycerol kinase (PGK) promoter from
any source that will be active or can easily be activated in the cell
into which it is inserted; however, the marker gene need not have its own
promoter attached, as it may be transcribed using the promoter of the
gene to be knocked out. In addition, the marker gene will normally have a
polyA sequence attached to its 3' end; this sequence serves to terminate
transcription of the marker gene. Preferred marker genes are any
antibiotic resistance gene such as neo (the neomycin resistance gene) and
beta-gal (beta-galactosidase).

[0083] After the eEF2 kinase genomic DNA fragment, cDNA molecule, or PCR
fragment has been digested with the appropriate restriction enzyme(s),
the marker gene molecule can be ligated with the native genomic DNA or
cDNA molecule using methods well known to the skilled artisan and
described in Sambrook et al., supra. In some cases, it will be preferable
to insert the marker sequence in the reverse or antisense orientation
with respect to the eEF2 kinase nucleic acid sequence; this reverse
insertion is preferred where the marker gene is operably linked to a
particularly strong promoter.

[0084] The ends of the DNA molecules to be ligated must be compatible;
this can be achieved by either cutting all fragments with those enzymes
that generate compatible ends, or by blunting the ends prior to ligation.
Blunting can be done using methods well known in the art, such as for
example by the use of Klenow fragment (DNA polymerase I) to fill in
sticky ends. After ligation, the ligated constructs can be screened by
selective restriction endonuclease digestion to determine which
constructs contain the marker sequence in the desired orientation.

[0085] The ligated DNA knockout construct may be transfected directly into
embryonic stem cells (discussed below), or it may first be placed into a
suitable vector for amplification prior to insertion. Preferred vectors
are those that are rapidly amplified in bacterial cells such as the
pBluescript II SK vector (Stratagene, San Diego, Calif.) or pGEM7
(Promega Corp., Madison, Wis.).

[0086] The eEF2 kinase knockout construct is typically transfected into
stem cells derived from an embryo (embryonic stem cells, or "ES cells").
ES cells are undifferentiated cells that are capable of taking up
extra-chromosomal DNA and incorporating it into their chromosomal DNA.
Generally, the ES cells used to produce the knockout mammal will be of
the same species as the knockout mammal to be generated. Thus for
example, mouse embryonic stem cells will usually be used for generation
of knockout mice.

[0087] The embryonic stem cell line used is typically selected for its
ability to integrate into and become part of the germ line of a
developing embryo so as to create germ line transmission of the knockout
construct. Thus, any ES cell line that is believed to have this
capability is suitable for use herein. Preferred ES cell lines for
generating knockout mice are murine cell lines D3 and E14 (American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852-1776 USA,
catalog nos. CRL 1934 and CRL 1821, respectively), or RW4 (Genome
Systems, Inc., 8620 Pennell Drive, St. Louis, Mich. 63114 USA, catalog
No. ESVJ-1182). The cells are cultured and prepared for DNA insertion
using methods well known to the skilled artisan such as those set forth
by Robertson (in: Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, E. J. Robertson, ed. IRL Press, Washington, D.C. (1987)), by
Bradley et al. (Current Topics in Devel. Biol., 20:357-371 (1986)) and by
Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1986)).

[0088] Insertion (also termed "transfection") of the knockout construct
into the ES cells can be accomplished using a variety of methods well
known in the art including for example, electroporation, microinjection,
and calcium phosphate treatment (see Lovell-Badge, in Robertson, ed.,
supra). A preferred method of insertion is electroporation.

[0089] The eEF2 kinase knockout construct DNA molecules to be transfected
into the cells can first be linearized if the knockout construct has
previously been inserted into a circular vector. Linearization can be
accomplished by digesting the DNA with a suitable restriction
endonuclease selected to cut only within the vector sequence and not
within the knockout construct sequence.

[0090] The isolated eEF2 kinase knockout construct DNA can be added to the
ES cells under appropriate conditions for the insertion method chosen.
Where more than one construct is to be introduced into the ES cells, the
DNA molecules encoding each construct can be introduced simultaneously or
sequentially. Optionally, homozygous eEF2 kinase knockout ES cells may be
generated by adding excessive eEF2 kinase knockout construct DNA to the
cells, or by conducting successive rounds of transfection in an attempt
to achieve homologous recombination of the knockout construct on both
endogenous eEF2 kinase alleles.

[0091] If the ES cells are to be electroporated, the ES cells and knockout
construct DNA are exposed to an electric pulse using an electroporation
machine and following the manufacturer's guidelines for use. After
electroporation, the cells are typically allowed to recover under
suitable incubation conditions. The cells are then screened for the
presence of the knockout construct.

[0092] Screening the ES cells can be accomplished using a variety of
methods, but typically, one screens for the presence of the marker
sequence portion of the knockout construct. Where the marker gene is an
antibiotic resistance gene, the cells can be cultured in the presence of
an otherwise lethal concentration of antibiotic. Those cells that survive
have presumably integrated the knockout construct. If the marker gene is
other than an antibiotic resistance gene, a Southern blot of the ES cell
genomic DNA can be probed with a sequence of DNA designed to hybridize
only to the marker sequence. If the marker gene is a gene that encodes an
enzyme whose activity can be detected (e.g., beta-galactosidase), the
enzyme substrate can be added to the cells under suitable conditions, and
the enzymatic activity of the marker gene can be analyzed.

[0093] The knockout construct may integrate into several locations in the
ES cell genome, and may integrate into a different location in each
cell's genome, due to the occurrence of random insertion events; the
desired location of insertion is within the eEF2 kinase endogenous gene
sequence. Typically, less than about 1-10 percent of the ES cells that
take up the knockout construct will actually integrate the knockout
construct in the desired location. To identify those cells with proper
integration of the knockout construct, chromosomal DNA can be extracted
from the cells using standard methods such as those described by Sambrook
et al., supra. This DNA can then be probed on a Southern blot with a
probe or probes designed to hybridize to the knockout construct DNA
digested with (a) particular restriction enzyme(s). Alternatively, or
additionally, a specific genomic DNA sequence can be amplified by PCR
with probes specifically designed to amplify that DNA sequence such that
only those cells containing the knockout construct in the proper position
will generate DNA fragments of the proper size.

[0094] Accordingly, the present invention also relates to an isolated
cell, wherein the cell contains a disruption in the eEF2 kinase gene. The
cell can be any type of cell, including cells isolated from a non-human
animal that is homozygous or heterozygous for the disruption to the gene,
for example, mouse embryo fibroblasts (MEFs). In preferred embodiments
the cell is an undifferentiated cell. In particularly preferred
embodiments the undifferentiated cell is selected from the group
consisting of a stem cell, an embryonic stem cell, an oocyte and an
embryonic cell. In especially preferred embodiment, the cell comprises a
disruption of the eEF2 kinase gene which encodes a catalytic domain of
the eEF2 kinase polypeptide. In an especially preferred embodiment, the
disruption comprises a portion of an eEF2 kinase gene, wherein a portion
of the eEF2 kinase gene is replaced by a selectable marker. In a
preferred embodiment the selectable marker is a gene which encodes for a
polypeptide selected from the group consisting of thymidine kinase,
neomycin phosphotransferase and hygromycin B phosphotransferase. In an
especially preferred embodiment the portion of the eEF2 kinase gene which
is replaced comprises exon 7.

[0095] After suitable ES cells containing the knockout construct in the
proper location have been identified, the cells can be incorporated into
an embryo. Incorporation may be accomplished in a variety of ways. A
preferred method of incorporation of ES cells is by microinjection into
an embryo that is at the blastocyst stage of development. For
microinjection, about 10-30 cells are collected into a micropipet and
injected into a blastocyst to integrate the ES cell into the developing
blastocyst.

[0096] The suitable stage of development for the blastocyst is species
dependent, however for mice it is about 3.5 days. The blastocysts can be
obtained by perfusing the uterus of pregnant females. Suitable methods
for accomplishing this are known to the skilled artisan, and are set
forth for example by Bradley (in Robertson, ed., supra).

[0097] While any blastocyst of the right age/stage of development is
suitable for use, preferred blastocysts are male and have genes coding
for a coat color or other phenotypic marker that is different from the
coat color or other phenotypic marker encoded by the ES cell genes. In
this way, the offspring can be screened easily for the presence of the
knockout construct by looking for mosaic coat color or other phenotypic
marker (indicating that the ES cell is incorporated into the developing
embryo). Thus, for example, if the ES cell line carries the genes for
white fur, the embryo selected will preferably carry genes for black or
brown fur.

[0098] An alternate method of preparing an embryo containing ES cells that
possess the knockout construct is to generate "aggregation chimeras". A
morula of the proper developmental stage (about 21/2 days old for mice)
is isolated. The zona pellucida can be removed by treating the morula
with a solution of mild acid for about 30 seconds, thereby exposing the
"clump" of cells that comprise the morula. Certain types of ES cells such
as the R1 cell line for mice can then be co-cultured with the morula
cells, forming an aggregation chimera embryo of morula and ES cells.

[0099] A refinement of the aggregation chimera embryo method can be used
to generate an embryo comprised of essentially only those ES cells
containing the knockout construct. In this technique, a very early stage
zygote (e.g., a two-cell stage zygote for mice) is given a mild electric
shock. This shock serves to fuse the nuclei of the cells in the zygote
thereby generating a single nucleus that has two-fold (or more) the DNA
of a naturally occurring zygote of the same developmental stage. These
zygotic cells are excluded from the developing embryo proper, and
contribute only to forming accessory embryonic structures such as the
extra-embryonic membrane. Therefore, when ES cells are co-cultured with
the zygotic cells, the developing embryo is comprised exclusively of ES
cells.

[0100] After the ES cells have been incorporated, the aggregation chimera
or transfected embryo can be implanted into the uterus of a
pseudopregnant foster mother. While any foster mother may be used,
preferred foster mothers are typically selected for their ability to
breed and reproduce well, and for their ability to care for their young.
Such foster mothers are typically prepared by mating with vasectomized
males of the same species. The pseudopregnant stage of the foster mother
is important for successful implantation, and it is species dependent.
For mice, this stage is about 2-3 days pseudopregnant.

[0101] Offspring that are born to the foster mother may be screened
initially for mosaic coat color or other phenotype marker where the
phenotype selection strategy (such as coat color, as described above) has
been employed. In addition, or as an alternative, chromosomal DNA
obtained from tail tissue of the offspring may be screened for the
presence of the knockout construct using Southern blots and/or PCR as
described above. The offspring that are positive for the eEF2 kinase
knockout construct will typically be heterozygous, although some
homozygous knockouts may exist, and can typically be detected by visually
quantifying the amount of probe that hybridizes to the Southern blots.

[0102] If homozygous knockout mammals are desired, they can be prepared by
crossing those heterozygous offspring believed to carry the knockout
construct in their germ line to each other; such crosses may generate
homozygous knockout animals. If it is unclear whether the offspring will
have germ line transmission, they can be crossed with a parental or other
strain and the offspring screened for heterozygosity. Homozygotes may be
identified by Southern blotting of equivalent amounts of genomic DNA from
mammals that are the product of this cross, as well as mammals of the
same species that are known heterozygotes, and wild-type mammals. Probes
to screen the Southern blots for the presence of the knockout construct
in the genomic DNA can be designed as set forth above.

[0103] Other means of identifying and characterizing the knockout
offspring are also available. For example, Northern blots can be used to
probe mRNA obtained from various tissues of the offspring for the
presence or absence of transcripts encoding either the gene knocked out,
the marker gene, or both. In addition, Western blots can be used to
assess the level of expression of the gene knocked out in various tissues
of these offspring by probing the Western blot with an antibody against
the protein encoded by the gene knocked out, or an antibody against the
marker gene product, where this gene is expressed. Finally, in situ
analysis (such as fixing the cells and labeling with antibody) and/or
FACS (fluorescence activated cell sorting) analysis of various cells from
the offspring can be conducted using suitable antibodies to look for the
presence or absence of the knockout construct gene product.

[0104] Both the heterozygous and homozygous eEF2 kinase knockout mammals
of this invention will have a variety of uses, since eEF2 kinase has been
implicated in regulation increased life span and increased resistance to
cell damage.

[0105] A functional knockout may also be achieved by the introduction of
an anti-sense construct that blocks expression of eEF2 kinase.

Compositions

[0106] The present invention also provides biologically compatible, cell
damage-inhibiting compositions comprising an effective amount of one or
more compounds identified as eEF2 kinase inhibitors, and/or the
expression-inhibiting agents as described hereinabove. In certain
aspects, the invention relates to a pharmaceutical composition for the
treatment or prevention of a condition involving cell damage or a
susceptibility to cell damage, comprising a therapeutically effective
amount of a compound that decreases phosphorylation of eEF2 by eEF2
kinase. In another aspect, the compound includes a polynucleotide
comprising a nucleotide sequence complementary to a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:
2.

[0107] The term "effective amount" or "therapeutically effective amount"
means that amount of a compound or agent that will elicit the biological
or medical response of a subject that is being sought by a medical doctor
or other clinician.

[0108] A biologically compatible composition is a composition, that may be
solid, liquid, gel, or other form, in which the compound, polynucleotide,
vector, and antibody of the invention is maintained in an active form,
e.g., in a form able to effect a biological activity. For example, a
compound of the invention would have inverse agonist or antagonist
activity on the eEF2 kinase; a nucleic acid would be able to replicate,
translate a message, or hybridize to a complementary mRNA of a eEF2
kinase; a vector would be able to transfect a eEF2 kinase cell and
expression the antisense, antibody, ribozyme or siRNA as described
hereinabove; an antibody would bind a eEF2 kinase polypeptide domain.

[0109] A preferred biologically compatible composition is an aqueous
solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer,
containing salt ions. Usually the concentration of salt ions will be
similar to physiological levels. Biologically compatible solutions may
include stabilizing agents and preservatives. In a more preferred
embodiment, the biocompatible composition is a pharmaceutically
acceptable composition. Such compositions can be formulated for
administration by topical, oral, parenteral, intranasal, subcutaneous,
and intraocular, routes. Parenteral administration is meant to include
intravenous injection, intramuscular injection, intraarterial injection
or infusion techniques. The composition may be administered parenterally
in dosage unit formulations containing standard, well-known non-toxic
physiologically acceptable carriers, adjuvants and vehicles as desired.

[0110] The term "carrier" means a non-toxic material used in the
formulation of pharmaceutical compositions to provide a medium, bulk
and/or useable form to a pharmaceutical composition. A carrier may
comprise one or more of such materials such as an excipient, stabilizer,
or an aqueous pH buffered solution. Examples of physiologically
acceptable carriers include aqueous or solid buffer ingredients including
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions such as
sodium; and/or nonionic surfactants such as TWEEN®, polyethylene
glycol (PEG), and PLURONICS®.

[0111] A particularly preferred embodiment of the present composition
invention is a cell damage-inhibiting pharmaceutical composition
comprising a therapeutically effective amount of an expression-inhibiting
agent as described hereinabove, in admixture with a pharmaceutically
acceptable carrier. Another preferred embodiment is a pharmaceutical
composition for the treatment or prevention of a condition related to
cell damage, or a susceptibility to the condition, comprising an
effective cell damage-inhibiting amount of a eEF2 kinase antagonist or
inverse agonist, its pharmaceutically acceptable salts, hydrates,
solvates, or prodrugs thereof in admixture with a pharmaceutically
acceptable carrier.

[0112] The term "pharmaceutically acceptable salts" refers to the
non-toxic, inorganic and organic acid addition salts, and base addition
salts, of compounds of the present invention. These salts can be prepared
in situ during the final isolation and purification of compounds useful
in the present invention.

[0113] The term "solvate" means a physical association of a compound
useful in this invention with one or more solvent molecules. This
physical association includes hydrogen bonding. In certain instances the
solvate will be capable of isolation, for example when one or more
solvent molecules are incorporated in the crystal lattice of the
crystalline solid. "Solvate" encompasses both solution-phase and isolable
solvates. Representative solvates include hydrates, ethanolates and
methanolates.

[0114] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in the
art in dosages suitable for oral administration. Such carriers enable the
pharmaceutical compositions to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for
ingestion by the patient. Pharmaceutical compositions for oral use can be
prepared by combining active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of granules,
after adding suitable auxiliaries, if desired, to obtain tablets or
dragee cores. Suitable excipients are carbohydrate or protein fillers,
such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch
from corn, wheat, rice, potato, or other plants; cellulose, such as
methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethyl-cellulose; gums including arabic and tragacanth; and
proteins such as gelatin and collagen. If desired, disintegrating or
solubilizing agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium
alginate. Dragee cores may be used in conjunction with suitable coatings,
such as concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or
dragee coatings for product identification or to characterize the
quantity of active compound, i.e., dosage.

[0115] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules made
of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules
can contain active ingredients mixed with filler or binders, such as
lactose or starches, lubricants, such as talc or magnesium stearate, and,
optionally, stabilizers. In soft capsules, the active compounds may be
dissolved or suspended in suitable liquids, such as fatty oils, liquid,
or liquid polyethylene glycol with or without stabilizers.

[0116] Preferred sterile injectable preparations can be a solution or
suspension in a non-toxic parenterally acceptable solvent or diluent.
Examples of pharmaceutically acceptable carriers are saline, buffered
saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,
potassium; calcium or magnesium chloride, or mixtures of such salts),
Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and
combinations thereof 1,3-butanediol and sterile fixed oils are
conveniently employed as solvents or suspending media. Any bland fixed
oil can be employed including synthetic mono- or di-glycerides. Fatty
acids such as oleic acid also find use in the preparation of injectables.

[0117] The composition medium can also be a hydrogel, which is prepared
from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as
a hydrophilic polyacrylic acid polymer that can act as a drug absorbing
sponge. Certain of them, such as, in particular, those obtained from
ethylene and/or propylene oxide are commercially available. A hydrogel
can be deposited directly onto the surface of the tissue to be treated,
for example during surgical intervention.

[0118] Embodiments of pharmaceutical compositions of the present invention
comprise a replication defective recombinant viral vector encoding the
polynucleotide inhibitory agent of the present invention and a
transfection enhancer, such as poloxamer. An example of a poloxamer is
Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.)
and is a non-toxic, biocompatible polyol. A poloxamer impregnated with
recombinant viruses may be deposited directly on the surface of the
tissue to be treated, for example during a surgical intervention.
Poloxamer possesses essentially the same advantages as hydrogel while
having a lower viscosity.

[0120] Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semi-permeable matrices of
solid hydrophobic polymers containing the antibody, which matrices are in
the form of shaped articles, e.g. films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides
(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON
DEPO®. (injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic
acid enable release of molecules for over 100 days, certain hydrogels
release proteins for shorter time periods. When encapsulated antibodies
remain in the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37° C., resulting in a loss of
biological activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thio-disulfide interchange,
stabilization may be achieved by modifying sulfhydryl residues,
lyophilizing from acidic solutions, controlling moisture content, using
appropriate additives, and developing specific polymer matrix
compositions.

[0121] As defined above, therapeutically effective dose means that amount
of protein, polynucleotide, peptide, or its antibodies, agonists or
antagonists, which ameliorate the symptoms or condition. Therapeutic
efficacy and toxicity of such compounds can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals, e.g.,
ED50 (the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio of toxic
to therapeutic effects is the therapeutic index, and it can be expressed
as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large
therapeutic indices are preferred. The data obtained from cell culture
assays and animal studies is used in formulating a range of dosage for
human use. The dosage of such compounds lies preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the dosage
form employed, sensitivity of the patient, and the route of
administration.

[0122] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays or in animal models,
usually mice, rabbits, dogs, or pigs. The animal model is also used to
achieve a desirable concentration range and route of administration. Such
information can then be used to determine useful doses and routes for
administration in humans. The exact dosage is chosen by the individual
physician in view of the patient to be treated. Dosage and administration
are adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors which may be taken into
account include the severity of the disease state, age, weight and gender
of the patient; diet, desired duration of treatment, method of
administration, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long acting pharmaceutical compositions might be administered
every 3 to 4 days, every week, or once every two weeks depending on
half-life and clearance rate of the particular formulation.

[0123] The pharmaceutical compositions according to this invention may be
administered to a subject by a variety of methods. They may be added
directly to tissues, complexed with cationic lipids, packaged within
liposomes, or delivered to eEF2 kinase-expressing cells by other methods
known in the art. Localized administration to the desired tissues may be
done by direct injection, transdermal absorption, catheter, infusion pump
or stent. The DNA, DNA/vehicle complexes, or the recombinant virus
particles are locally administered to the site of treatment. Alternative
routes of delivery include, but are not limited to, intravenous
injection, intramuscular injection, subcutaneous injection, aerosol
inhalation, oral (tablet or pill form), topical, systemic, ocular,
intraperitoneal and/or intrathecal delivery. Examples of ribozyme
delivery and administration are provided in Sullivan et al. WO 94/02595.

[0124] Antibodies according to the invention may be delivered as a bolus
only, infused over time or both administered as a bolus and infused over
time. Those skilled in the art may employ different formulations for
polynucleotides than for proteins. Similarly, delivery of polynucleotides
or polypeptides will be specific to particular cells, conditions,
locations, etc.

[0125] As discussed hereinabove, recombinant viruses may be used to
introduce DNA encoding polynucleotide agents useful in the present
invention. Recombinant viruses according to the invention are generally
formulated and administered in the form of doses of between about
104 and about 1014 pfu. In the case of AAVs and adenoviruses,
doses of from about 106 to about 1011 pfu are preferably used.
The term pfu ("plaque-forming unit") corresponds to the infective power
of a suspension of virions and is determined by infecting an appropriate
cell culture and measuring the number of plaques formed. The techniques
for determining the pfu titre of a viral solution are well documented in
the prior art.

[0126] The polypeptides or the polynucleotides employed in the methods of
the present invention may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. To perform
the methods it is feasible to immobilize either the polypeptide of the
present invention or the compound to facilitate separation of complexes
from uncomplexed forms of the polypeptide, as well as to accommodate
automation of the assay. Interaction (e.g., binding of) of the
polypeptide of the present invention with a compound can be accomplished
in any vessel suitable for containing the reactants. Examples of such
vessels include microtitre plates, test tubes, and microcentrifuge tubes.
In one embodiment, a fusion protein can be provided which adds a domain
that allows the polypeptide to be bound to a matrix. For example, the
polypeptide of the present invention can be "His" tagged, and
subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions
with the polypeptides of the present invention can be adsorbed to IgG,
which are then combined with the cell lysates (e.g., (35S-labelled)
and the candidate compound, and the mixture incubated under conditions
favorable for complex formation (e.g., at physiological conditions for
salt and pH). Following incubation, the plates are washed to remove any
unbound label, and the matrix is immobilized. The amount of radioactivity
can be determined directly, or in the supernatant after dissociation of
the complexes. Alternatively, the complexes can be dissociated from the
matrix, separated by SDS-PAGE, and the level of the protein binding to
the protein of the present invention quantitated from the gel using
standard electrophoretic techniques.

[0127] Other techniques for immobilizing protein on matrices can also be
used in the method of identifying compounds. For example, either the
polypeptide of the present invention or the compound can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated protein
molecules of the present invention can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art (e.g.,
biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in
the wells of streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with the polypeptides of the present
invention but which do not interfere with binding of the polypeptide to
the compound can be derivatized to the wells of the plate, and the
polypeptide of the present invention can be trapped in the wells by
antibody conjugation. As described above, preparations of a labeled
candidate compound are incubated in the wells of the plate presenting the
polypeptide of the present invention, and the amount of complex trapped
in the well can be quantitated.

EXAMPLES

[0128] Using a mouse knockout model system, it has been determined that
loss of eEF2 kinase activity increases protein synthesis and degradation
rates and reduces damage to a cell or increases resistance to damage to a
cell. Mice lacking a functional eEF2 kinase appear and have normal
development, behavior and reproduction.

Example 1

Plasmids, Antibodies and Cells

[0129] Retroviral vector used for eEF2 kinase overexpression is
constructed by subcloning of eEF2 kinase cDNA from the pSIT retroviral
vector7 into LXSN vector (Clontech) using Eco RI/XhoI cloning sites.
Stable cell line is prepared through infection of MEFs with pBabe-neo
retroviral vector containing SV-40 large T antigen (a kind gift from Dr.
J. Yuan). GSE 56 cell lines are established using retroviral vector LXSP,
containing GSE5612. LXSP vector is prepared from LXSN vector by the
substitution of neomycin marker with puromycin. Antibodies against p21
(F5) and p53 (Ab-1) are from Calbiochem Inc.; antibodies against eEF2 and
phospho-eEF2 are from Cell Signaling Inc. eEF2 kinase and eEF2
kinase.sup.+/+ primary mouse embryonic fibroblasts used in this study are
isolated from 10-12 day embryos following standard protocols. Unless
indicated, all cell lines are maintained in DMEM with 10% fetal bovine
serum.

Example 2

Transfection and Retroviral Infection

[0130] Packaging cells (Phoenix line) are plated in 60-mm plates and
transfected with 5 μg of retroviral vector DNA using the standard
calcium phosphate procedure. Medium is changed after 8 hours.
Virus-containing medium supplemented with 8 μg of Polybrene (Sigma) is
collected at 24 and 48 hours post-transfection and used for infection.
Infected cells are selected for the resistance to an appropriate
selection agent.

[0133] MTT Survival Assay: MEFs used in this study (2×103 cells
per well of a 96 well plate) are incubated in the presence of indicated
drugs for 24 hours. Cell viability is determined using the standard MTT
assay30. Each experiment is repeated three times for each drug and
each cell line using three parallel wells for each drug concentration.
For colony formation assay cells are treated with indicated drugs for 24
hours and replated with complete DMEM at low density (500 cells per well
in 6-well plate) in duplicate. After 10 days colonies are stained with 10
mg/ml methylene blue (Sigma Inc.) in 50% methanol.

[0135] In order to investigate the function of eEF2 kinase an eEF2 kinase
knockout mouse is generated by disrupting the eEF2 kinase gene in mouse
embryonic stem (ES) cells. A targeting vector is used, in which exon 7
and the majority of exon 8 are replaced with the neomycin resistance gene
(FIG. 1a), resulting in the elimination of a portion of the catalytic
domain of eEF2 kinase (FIG. 1a, b; FIG. 2A, 2B). eEF2 kinase.sup.+/- ES
cell clones are identified and used to obtain chimeras and subsequently
eEF2k.sup.+/- mice. Mating of eEF2 kinase.sup.+/- mice produced progeny
of eEF2 kinase.sup.+/-, eEF2 kinase.sup.+/+ and eEF2 kinase.sup.-/- mice
with the expected ratio of 2:1:1. No phosphorylated eEF2 is observed in
the tissue extracts of the eEF2 kinase.sup.-/- mice, indicating their
complete lack of eEF2 kinase activity (FIG. 1c). The knockout mice are
viable, do not have visible abnormalities and give normal progeny for
many generations.

[0136] eEF2 kinase is a Ca2±/calmodulin-dependent enzyme whose
cellular activity is previously demonstrated to increase upon incubation
with Ca2+ ionophores ionomycin or A23187, which can induce apoptotic
cell death. The possible correlation between activation of eEF2 kinase
and the resultant phosphorylation of eEF2 and Ca2+ ionophore-induced
cell death is addressed by comparing the viability of wild type and eEF2
kinase knockout mouse embryonic fibroblasts (MEFs) after incubation of
each with different concentrations of ionomycin. As shown in FIG. 1d,
exposure of eEF2 kinase.sup.+/+ MEFs to increasing concentrations of
ionomycin from 1 to 10 μM resulted in a progressive decrease of cell
viability. Notably, approximately 70% of cells die after incubation for
24 h with 10 μM ionomycin, whereas the same concentrations of
ionomycin have virtually no effect on the viability of eEF2
kinase.sup.-/- MEFs. These results suggest that activation of eEF2 kinase
may facilitate cell death induced by ionomycin and that inactivation of
eEF2 kinase results in an increased resistance to damage caused by
Ca2+ ionophores.

[0137] eEF2 kinase can also be activated by acidic pH. Acidic pH is
cytotoxic and therefore activation of eEF2 kinase by acidic pH can be
involved in the regulation of cell death. The effect of acidic pH on the
viability of eEF2 kinase knockout and wild types MEFs is also analyzed.
FIG. 1e shows that eEF2 kinase.sup.-/- MEFs are significantly more
resistant than wild type MEFs to the cytotoxic effect of acidic pH. The
increased viability of eEF2 kinase knockout cells in an acidic
environment is also observed in the stable cell lines derived from MEFs.
Using a low-density clonogenic assay it is shown that after incubation at
pH 6 for 3 hours, eEF2 kinase knockout cells produce significantly more
colonies than wild type cells (FIG. 1g). eEF2 kinase.sup.-/- MEFs are
also significantly more resistant than wild type MEFs to hydrogen
peroxide (FIG. 1f). Thus, these results demonstrate that knockout of eEF2
kinase increases resistance of cells to stress, induced by Ca2+
ionophore and acidic pH, as well as to oxidative stress induced by
hydrogen peroxide.

Example 8

Sensitivity to Chemotherapeutic Drugs

[0138] Next the sensitivity of knockout and wild type MEFs to the
chemotherapeutic drugs camptothecin (CPT) and doxorubicin (DOX) is
analyzed. As shown in FIGS. 3a and 3b, eEF2 kinase.sup.-/- MEFs and their
stable cell lines are significantly more resistant than their wild type
counterparts to CPT and DOX, respectively. Introduction of eEF2 kinase
cDNA into eEF2 kinase knockout cells restored sensitivity of these cells
to DOX to the level observed in wild type cells (FIG. 3c).

[0139] To investigate the mode of cell death that is affected by eEF2 an
annexin V assay and DNA fragmentation analysis is performed. As shown in
FIG. 3d the percentage of apoptotic annexin V positive cells after 24
hours of treatment with doxorubicin is significantly lower in eEF2 kinase
cells than in eEF2 kinase.sup.+/+ cells. Apoptotic DNA ladder formation
in wild type and eEF2 kinase knockout cells in response to serum
starvation is also analyzed. Incubation of eEF2 kinase.sup.+/+ cells in
serum free media for 48 h resulted in significant DNA fragmentation,
whereas no DNA ladder formation is observed in eEF2 kinase.sup.-/- cells
incubated under the same conditions (FIG. 3e). This suggests that the
absence of eEF2 kinase results in inhibition of apoptosis and therefore
eEF2 kinase might be a factor that facilitates apoptosis.

[0140] Since the tumor suppressor p53 is known to be involved in
modulating the sensitivity of MEFs to various cytotoxic drugs and
induction of apoptosis, it is determined whether the effects of the eEF2
kinase knockout on cell sensitivity to doxorubicin might depend on
functional p53. Sensitivity to DOX is assessed in wild type and eEF2
kinase knockout MEFs in which p53 is inactivated by overexpression of GSE
56, a carboxyl-terminal portion of p53 that acts as a dominant negative
p53 mutant, or by incubation with pifithrin α (PFT α), a
chemical p53 inhibitor. Inactivation of p53 either by GSE56 or PFTα
only slightly affected drug sensitivity of eEF2 kinase knockout
fibroblasts, while strongly decreasing sensitivity of wild type cells
(FIG. 4a, b), suggesting that the effect of eEF2 kinase on drug
sensitivity depends on functional p53. Additionally, the effect of DOX
treatment on the induction of p53 and cyclin-dependent kinase inhibitor
p21 (WAF1), whose expression is known to be regulated by p53, is
determined. The expression of p53 is undetectable in both untreated eEF2
kinase.sup.-/- and untreated wild type MEFs. After treatment with 600
ng/ml of DOX for 24 h, p53 is similarly induced in both eEF2
kinase.sup.-/- and eEF2 kinase.sup.+/+ MEFs. However, p21 WAF1 is induced
significantly more strongly in eEF2 kinase.sup.+/+ MEFs (FIGS. 3c and
3d). Expression of several p53 dependent genes in eEF2 kinase knockout
and wild type cells is also determined after treatment with doxorubicin
using RT-PCR. In addition to p21 WAF1, the induction of apoptosis-related
genes, GADD45 and PIG3 is significantly higher in eEF2 kinase.sup.+/+ DOX
treated cells than in eEF2 kinase.sup.-/- DOX treated cells. These
results suggest that although induction of p53 in eEF2 kinase knockout
cells is comparable to that of the wild type, its transactivation
activity is altered.

[0141] Long-term survival assays in eEF2 kinase.sup.+/-, eEF2
kinase.sup.+/+ and eEF2 kinase.sup.-/- mice reveal that knockout of eEF2
kinase results in a significant increase in maximal life span (FIG. 5a,
b). Maximal lifespan, defined as the average age of the last 10% of
surviving mice, is increased by approximately 30% in eEF2 kinase.sup.-/-
mice (36.6 month) and approximately 18% in eEF2 kinase.sup.+/- mice (33.1
month) in comparison with eEF2 kinase.sup.+/+ mice (28 month). Since
increase in maximal life span is observed in both eEF2 kinase.sup.-/- and
eEF2 kinase.sup.+/- mice, the complete elimination of eEF2 kinase is not
required for the life span extending effect. The significant increase in
maximal life span in eEF2 kinase knockout mice is not accompanied by an
increase in median life span. Maximal life span is considered to be a key
parameter in the measurement of longevity and its extension indicates a
genuine slowing of the aging process. In contrast to maximal life span,
which depends on the cumulative effect of many different factors related
to aging, median life span is often determined by a single factor, that
causes death in the majority of animals in the population and which may
or may not be related to aging. Therefore the increase in maximal, but
not median life span in eEF2 kinase.sup.-/- and eEF2 kinase.sup.+/- mice
suggests that the decrease of eEF2 kinase affects aging per se.

[0142] The increased maximal life span in eEF2 kinase knockout mice can be
related to increased cellular stress resistance. In addition there is a
correlation between life span of various mammalian species and stress
resistance of fibroblasts derived from them.

[0143] Increased maximal life span in eEF2 kinase knockout mice can also
be related to altered regulation of p53 and p21. Increased activation of
p53 is known to cause premature aging in mice and overexpression of p21
results in the induction of various genes associated with senescence and
aging, including p66SHC. Therefore, the reduced activation of p53
observed in eEF2 kinase.sup.-/- cells and the reduced induction of p21
that it leads to, can contribute to increased longevity in eEF2 kinase
knockout mice.

Example 9

Sensitivity to Radiation

[0144] Gamma irradiation of mice: 8 to 12 week old mice are irradiated at
a dose of 8 grays (Gy) of whole-body gamma irradiation produced by
Caesium-137 source (Nordiom gammacell 40). Each cohort of mice consists
of 10 mice including 5 males and 5 females.

[0145] Preparation of MEFs: Mouse embryonic fibroblasts (MEFs) are
prepared from E13.5 embryos and immortalized by large T antigen via
retrovirus infection. Virus is collected from the medium of transient
triple-transfected 239T cells by three plasmids including VSV, gal/pol,
and pBebe-neo TcDNA.

[0146] TUNEL assay: Cells are treated with or without 1.6 μM of
doxorubicin for 24 hours. After treatment, cells are collected and fixed
in 1% paraformaldehyde for 15 minutes on ice. Cells are stored in 75%
ethanol at -20° C. until staining, which is performed according to
the manufacturer's instructions (In Situ Cell Death Detection kit,
Roche). Apoptotic cells are labeled with fluorescein and counted by flow
cytometry.

[0147] The effect of eEF2 kinase deficiency on the short-term survival of
mice under stress is analyzed. Mice are irradiated with 8 Gy of
whole-body γ-irradiation. After irradiation, 50% of wild type mice
die within 16 days; however none of the eEF2K-deficient mice die during
the same interval (FIG. 6a). Within one month after irradiation the hair
color of surviving, normally black wild type mice turns grey but,
unexpectedly, this does not occur in eEF2 kinase deficient mice (FIG.
6b). In addition, significant hair loss is noted in irradiated wild type
mice, but not in irradiated eEF2K-/- mice.

[0148] Because γ-irradiation is known to induce apoptosis, the
increased resistance to γ-irradiation observed in eEF2K-deficient
mice may be due to a corresponding increase in the resistance of
eEF2K-deficient cells to apoptosis. To test this possibility, the effect
of eEF2K deficiency on apoptosis in cells isolated from eEF2K-/- mice is
analyzed. Cells from eEF2K deficient mice are significantly more
resistant to apoptosis induced by doxorubicin or hydrogen peroxide. As
can be seen in FIG. 7a, significant cell death is observed in wild type
mouse embryonic fibroblasts (MEFs) treated for 24 hours with 1.6 μM
doxorubicin. However, much less cell death is observed in eEF2K-deficient
cells treated in the same manner. The results of the TUNEL assay suggest
that the reduction in cell death in eEF2K-deficient cells is due to
decreased apoptosis (FIG. 7b). To verify that the decreased apoptosis is
due to the absence of eEF2K, eEF2K cDNA is introduced into
eEF2K-deficient MEFs. As can be seen in FIG. 7c, after treatment with
hydrogen peroxide or doxorubicin, MEFs carrying eEF2K cDNA have more
activated caspase 3 than eEF2K-deficient cells from which they are
derived, thus confirming that eEF2K enhances apoptosis.

[0150] Using antibody that specifically recognizes phosphorylated eEF2,
the distribution of phosphorylated eEF2 in tissue culture cells and in
various human tissues is analyzed. Western blot analysis reveals strong
and persistent phosphorylation of eEF2 in NIH3T3 cells treated with
hydrogen peroxide for various time periods (FIG. 8a). Immunocytochemical
analysis of these cells show that levels of phosphorylated eEF2 are high
in rounding cells and highest in cells undergoing apoptosis, whereas no
significant phosphorylation of eEF2 is detected in control cells grown
under standard conditions (FIG. 8b). Similarly, treatment of HeLa cells
with hydrogen peroxide results in a dramatic and selective increase in
phosphorylation of eEF2 in apoptotic cells with condensed chromatin (FIG.
8c).

[0151] Various human tissues are also examined for the presence of
phosphorylated eEF2 using human multiple tissue arrays. Phosphorylated
eEF2 is not detectable in most tissues. However, significant
phosphorylation of eEF2 in lymph nodes is observed (FIGS. 8d-e).
Particularly intense phosphorylation is found in macrophages that likely
represent staining of phagocytized lymphocytes undergoing apoptosis (FIG.
8e). Phosphorylated eEF2 is also detected in pyramidal neurons in
histological sections of Alzheimer's disease brains (FIG. 80, but not in
neurons of neurologically normal, age-matched control brains (FIG. 8g).
These results suggest that activation of eEF2 kinase is associated with
the cellular response to stress and cell death.

[0152] The foregoing examples and description of the preferred embodiments
should be taken as illustrating, rather than as limiting the present
invention as defined by the claims. As will be readily appreciated,
numerous variations and combinations of the features set forth above can
be utilized without departing from the present invention as set forth in
the claims. Such variations are not regarded as a departure from the
spirit and script of the invention, and all such variations are intended
to be included within the scope of the following claims.